title: Production of human normal adult and fetal hemoglobins in Escherichia coli.
abstract: A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.
authors.affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
authors.forename: T J
authors.initials: TJ
authors.lastname: Shen
authors.forename: N T
authors.initials: NT
authors.lastname: Ho
authors.forename: M
authors.initials: M
authors.lastname: Zou
authors.forename: D P
authors.initials: DP
authors.lastname: Sun
authors.forename: P F
authors.initials: PF
authors.lastname: Cottam
authors.forename: V
authors.initials: V
authors.lastname: Simplaceanu
authors.forename: M F
authors.initials: MF
authors.lastname: Tam
authors.forename: D A
authors.initials: DA
authors.lastname: Bell
authors.forename: C
authors.initials: C
authors.lastname: Ho
mesh_terms: D000328:Adult; D000626:Aminopeptidases / Q000235:genetics; D004926:Escherichia coli; D005319:Fetal Hemoglobin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006441:Hemoglobin A / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008706:Methemoglobin / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D008706:Methemoglobin; D006441:Hemoglobin A; D005319:Fetal Hemoglobin; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; D010100:Oxygen